The quantitative determination of the bioavailability (BA) of a topically applied drug at its site of action in the skin represents an unmet regulatory need. The majority of dermatological drug targets are found in the epidermis and upper dermis (including appendages such as hair follicles and sebaceous glands), beneath the principal skin barrier, the stratum corneum (SC). While some alternative methods to clinical endpoint BA and bioequivalence (BE) studies have been identified, the evaluation of other surrogate approaches is a ?work-in-progress?. The central hypothesis of the research strategy proposed is that spectroscopic (in particular, Raman) imaging is able to provide a non-invasive, accurate, sensitive and reproducible determination of the rate and extent to which a topically administered drug becomes available at its site of action below the SC (and, specifically, the viable epidermis). The initial goal is to establish experimental methods, combining Raman and mass spectrometry imaging, to prove this hypothesis ex vivo; subsequently, the objective is to develop Raman imaging in vivo, and to calibrate the approach using complementary techniques. The project has the following specific aims: (a) to conceive strategies by which unambiguous Raman spectroscopic analysis of a drug in the skin can be achieved despite the potential for significant, background signal interference; (b) to evolve a robust approach to correct for drug signal attenuation as a function of increasing depth of measurement in the skin; (c) to demonstrate that continuous, real- time Raman imaging after topical application of a formulation can measure the drug?s ?input kinetics? into the viable epidermis ex vivo; (d) to correlate and validate Raman spectroscopic assessment of drug input with parallel, high-sensitivity mass spectroscopic analysis and imaging; (e) to develop Raman imaging to quantify drug input kinetics into the viable epidermis in vivo and to calibrate the method using SC sampling and reverse iontophoresis, another non-invasive method, in combination with mathematical modelling; (f) to demonstrate that the refined spectroscopic tool can characterise the epidermal BA of a topically applied drug and distinguish correctly between formulations that are BE and those that are not; and (g) to apply spatio-temporal mathematical modelling to identify the mechanistic processes underlying the observed results, to explore the impact of drug properties on those mechanisms, and to estimate and validate key metrics of topical bioavailability. In summary, therefore, the proposed project combines different, but synergistic, approaches to assess topical BA/BE with novel spectroscopic imaging strategies to demonstrate that the routine, facile and noninvasive measurement of drug pharmacokinetics in the skin in vivo is an attainable objective with considerable potential for application in regulatory science and decision-making.